KR20170010072A - Production of squalene using yeast - Google Patents

Abstract

The present invention provides compositions and methods for producing isoprenoids, including squalene. In certain aspects and embodiments, genetically modified yeast and uses thereof are provided. In some aspects and embodiments, the genetically modified yeast produces an isoprenoid, preferably squalene. The expression or activity of enzymes involved in the production of squalene, such as acetyl-CoA carboxylase (or "ACCase"), HMG-CoA reductase, squalene epoxidase, and squalene synthase, . One or more genes of the genetically modified yeast may be modified by the gene repair oligonucleotide base. It also provides a method for producing squalene using genetically modified yeast. The present invention also provides squalene produced by genetically modified yeast.

Description

Production of squalene using yeast {PRODUCTION OF SQUALENING USING YEAST}

Cross-reference to related application

This patent application is a continuation-in-part of US patent application Ser. No. 61 / 055,931 entitled " Production of Squalene Using Yeast " filed on May 23, 2008 (the disclosure of which is hereby incorporated by reference herein for all purposes) ).

Field of the Invention

Methods and compositions for producing isoprenoids, such as, for example, squalene, using yeast are provided.

It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and it is not intended to be exhaustive or to limit the invention to the precise forms disclosed.

For example, isoprenoids such as squalene are commercially important types of lipids. It has excellent lubricity and oxidation stability, low pour point, low freezing point, high flash point, and easy biodegradability. Squalene is currently produced at high unit prices by extraction from cold water shark liver oil or olive oil. Economically feasible applications of squalene and squalane (an all-hydrogenated derivative of squalene), due to their high unit cost, can be used as a chemical intermediate for clock lubricants, pharmaceuticals / nutrients, cosmetics, fragrances, .

However, there is a large potential market for biodegradable lubricants, lubricant additives, and hydraulic oils. The biodegradability of these products is particularly important in the case of environmentally sensitive applications, such as agricultural applications, where significant quantities of lubricants or hydraulic oils can be exposed to the environment. The potential market for biodegradable lubricants, lubricant additives, and hydraulic oils is fairly large, estimated at about 5 million metric tons per year.

Biodegradable lubricants, lubricant additives, and hydraulic oils derived from plant and animal fats and oils are available, but there are problems. It typically has a flash point (i. E., Decomposed or burned under normal high temperature engine conditions) that solidifies at relatively high temperatures (i.e., solidifies in cold weather) and is too low to be used under high temperature conditions.

Accordingly, there is a need for a cost effective squalene production process that enables large scale production of biodegradable lubricants, lubricant additives, and squalene and squalane in hydraulic oils, which can be widely used.

"A large amount of squalene and several polyunsaturated fatty acids" is produced in Chang et al. Lt; RTI ID = 0.0 > Pseudozyma sp. & Lt ; / RTI > JCC207. Chang et al. Described the isolation of Schizojima species JCC207 from seawater around Guam in the US and confirmed that the Schizochima species JCC207 is a new species, or P. regulosa or P. aphidis , Is uncertain as to the variation of the variation of In this article, "Squalene production rate of [Schizochima sp. JCC207] was investigated under different conditions".

Using Yeast Fermentation to Produce Cost-Effective Biodegradable Lubricants at http://statusreports.atp.nist.gov/reports/95-01-0148PDF.pdf (Using Yeast Fermentation to Produce Cost-Effective and Biodegradable Lubricants) Dow AgroSciences LLC (Dow AgroSciences LLC) "We are proposing genetic engineering to change the metabolic characteristics of persistent (oily) yeasts in order to increase the ability of yeast to produce isoprene through biosynthesis ([t] he company proposed to use genetic engineering to alter the metabolic characteristics of an oleaginous (oily) yeast to increase the ability of yeast to produce isoprenes through biosynthesis "). Specifically, four enzymes were targeted: ACCase, hydroxymethylglutaryl CoA reductase (HMGR), squalene synthetase, and squalene epoxidase.

U.S. Patent No. 5,460,949 discloses "a method for increasing the accumulation of squalene and certain sterols in yeast ([a] increasing the accumulation of squalene and specific sterols in yeast"). In particular, the accumulation of squalene and sterol is increased by increasing the expression level of a gene encoding a polypeptide having HMG-CoA reductase activity ([s] qualene and sterol accumulation is increased by increasing the expression level of a gene encoding a polypeptide having the HMG-CoA reductase activity ".

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for producing squalene from yeast.

In one aspect, there is provided a genetically modified yeast that produces an isoprenoid. In certain embodiments, the genetically modified yeast produces squalene.

In a related aspect, genetically modified to provide a genetically modified yeast that produces squalene at an increased level compared to the corresponding natural yeast. In certain embodiments of this aspect of the invention, the genetically modified yeast expresses one or more modified enzymes having one or more mutations. In certain embodiments of this aspect, the expression level of one or more enzymes in the genetically modified yeast is increased or decreased as compared to the corresponding natural yeast. In a related embodiment, the genetically modified yeast expresses one or more modified enzymes having one or more mutations, and the expression level of one or more enzymes in the genetically modified yeast is increased or decreased when compared to the corresponding natural yeast . In certain preferred embodiments, the genetically modified yeast, such as those provided herein, is genetically modified by introducing a mutation into the enzyme using a gene repair oligo base. In some embodiments, genetically modified yeast, such as those provided herein, can be prepared by coding an enzyme to increase or decrease the expression of the enzyme, as described in U.S. Patent Application Nos. 10 / 411,969 and 11 / 625,586 Genetically modified by introducing one or more mutations at or near the translation initiation site of the gene. In certain embodiments, modified enzymes in genetically modified yeasts such as those provided herein include, but are not limited to, acetyl-CoA carboxylase (or "ACCase"), HMG- CoA reductase, squalene epoxidase, squalene synthase, And an enzyme selected from the group consisting of citrate lyase.

The nucleobase base comprises a base, which is purine, pyrimidine, or a derivative or analogue thereof. A nucleoside is, for example, a nucleobase group comprising a pentosupuranosyl moiety, such as an optionally substituted riboside or 2'-deoxyribose. The nucleoside may be linked by one of several linking moieties which may or may not contain phosphorus. A nucleoside linked by an unsubstituted phosphodiester linkage is termed a nucleotide. As used herein, the term "nucleobase" includes peptide nucleobases, which are subunits of peptide nucleic acids, and morpholine nucleobases, as well as nucleosides and nucleotides.

Oligonucleobases are polymers of nucleobases, which can be hybridized by Watson-Crick base pairing with DNA having a complementary sequence. The oligonucleotide base chain has a single 5 ' and 3 ' ends which are the last nucleobases of the polymer. Certain oligonucleotide bases may comprise all types of nucleobases. Oligonucleobase compounds are compounds that are complementary and contain one or more oligonucleotide bases that hybridize by Watson-Crick base pairing. The nucleobase is deoxyribo- or ribo-type. A ribose-type nucleobase is a pentosupuranosyl comprising a nucleobase in which the 2 ' carbon is methylene substituted with hydroxyl, alkyloxy or halogen. Deoxyribo-type nucleobases are nucleobases other than the ribo-type nucleobases, including all nucleobases that do not contain the pentosporanosyl moiety.

The oligonucleotide base strand generally comprises an oligonucleotide base strand and a segment or region of an oligonucleotide base strand. The oligonucleotide base strand has a 3 ' end and a 5 ' end. When the oligonucleotide base strand occupies the same space as the strand, the 3 ' and 5 ' ends of the strand also become the 3 ' and 5 ' ends of the strand.

As used herein, the term "gene-repairing oligonucleotide base" refers to a nucleic acid comprising a hybrid double oligonucleotide, a molecule containing a non-nucleotide, a single strand oligodeoxynucleotide and other gene repair molecules described in detail below , ≪ / RTI > an oligonucleotide base.

In some embodiments, the genetically altered yeast, such as those provided herein, is derived from retentive yeast. In certain preferred embodiments, the genetically modified yeast as provided herein is selected from the group consisting of Cryptococcus curvatus , Yarrowia lipolytica , Rhodotorula glutinus , ≪ / RTI > Rhodosporidium toruloides . In some preferred embodiments, the genetically modified yeast is derived from a yeast selected from the group consisting of Cryptococcus cuvatus, Yarrowia polyethica, and Rhodotorula glutinus. In a related embodiment, the genetically modified yeast is derived from a yeast selected from the group consisting of Cryptococcus cactus, and Rhodotorula glutinus. In certain preferred embodiments, the genetically altered yeast is not from Yarrowia polyricca.

In certain preferred embodiments, the enzyme modified in a genetically modified yeast such as provided herein is an acetyl-CoA carboxylase (or "ACCase"). In some preferred embodiments, the acetyl-CoA carboxylase in the genetically modified yeast is reduced when compared to its natural activity and / or expression in the corresponding yeast; Activity and / or expression is extinguished. In other embodiments, the acetyl-CoA carboxylase can be modified to alter its substrate selectivity. In some preferred embodiments, the genetically modified yeast is modified such that the activity and / or expression of the acetyl-CoA carboxylase is reduced when compared to the corresponding natural yeast, and the activity is not abolished. In some preferred embodiments, the genetically modified yeast has an activity and / or expression of the acetyl-CoA carboxylase in genetically modified yeast of about 90% of the activity and / or expression of the corresponding natural yeast; Or about 80%; Or about 70%, or about 60%, or about 50%; Or about 40%; Or about 30%, or about 20%; Or about 10%, or about 5%. In a related embodiment, the genetically modified yeast has 90-95% of the activity and / or expression of the activity and / or expression of the acetyl-CoA carboxylase in the genetically modified yeast relative to that of the native yeast; Or 80-90%, or 70-80%; 60-70%; Or 50-60%; Or 40-50%; Or about 30-40%; Or about 20-30%; About 10-20%; Or about 5-10%, or about 2-5%.

In certain preferred embodiments, the enzyme modified in a genetically modified yeast such as provided herein is an HMG-CoA reductase. In some preferred embodiments, the HMG-CoA reductase in genetically modified yeast is modified such that its activity and / or expression is increased when compared to the corresponding natural yeast. In other embodiments, the HMG-CoA reductase can be modified to alter its substrate selectivity. In certain preferred embodiments, the genetically modified yeast is selected from the group consisting of: at least 1.2-fold of the activity and / or expression of the HMG-CoA reductase activity and / or expression of the corresponding natural yeast in the genetically altered yeast; Or 1.5-fold, or 2-fold; Or 3-fold, or 4-fold; Or 5-fold; Or 10-fold, or 10-fold; Or 20-fold; Or 50-fold; Or 100-fold, or 1,000-fold; Or 10,000-fold, or 100,000-fold, or 1,000,000-fold higher.

In certain preferred embodiments, the enzyme modified in a genetically modified yeast such as provided herein is a squalene epoxidase. In some preferred embodiments, the squalene epoxidase in the genetically modified yeast is modified such that its activity and / or its expression is reduced or the activity and / or expression are extinguished when compared to the corresponding natural yeast. In other embodiments, the squalene epoxidase can be modified to alter its substrate selectivity. In some preferred embodiments, the genetically modified yeast is modified such that the activity and / or expression of the squalene epoxidase is reduced when compared to the corresponding natural yeast, and the activity is not abolished. In some preferred embodiments, the genetically altered yeast has an activity and / or expression of the squalene epoxidase in the genetically modified yeast of about 90% of the activity and / or expression of the corresponding natural yeast; Or about 80%; Or about 70%; Or about 60%; Or about 50%; Or about 40%; Or about 30%; Or about 20%; Or about 10%; Or about 5%. In a related embodiment, the genetically modified yeast is selected from the group consisting of: 90-95% of the activity and / or expression of the squalene epoxidase in the genetically modified yeast relative to the activity and / or expression of the corresponding natural yeast; Or 80-90%; Or 70-80%; 60-70%; Or 50-60%; Or 40-50%; Or about 30-40%; Or about 20-30%; About 10-20%; Or about 5-10%; Or between about 2-5%.

In certain preferred embodiments, the enzyme modified in a genetically modified yeast such as provided herein is squalene synthase. In some preferred embodiments, the squalene synthase in the genetically modified yeast is modified such that its activity and / or expression is increased when compared to the corresponding natural yeast. In other embodiments, the squalene synthase may be modified to alter its substrate selectivity. In certain preferred embodiments, the genetically altered yeast is selected so that the activity and / or expression of squalene synthase in the genetically altered yeast is at least 1.2-fold of the activity and / or expression of the corresponding natural yeast; Or 1.5-fold, or 2-fold; Or 3-fold, or 4-fold; Or 5-fold; Or 10-fold, or 10-fold; Or 20-fold; Or 50-fold; Or 100-fold, or 1,000-fold; Or 10,000-fold, or 100,000-fold, or 1,000,000-fold higher.

In certain preferred embodiments, the enzyme modified in a genetically modified yeast such as provided herein is an ATP citrate lyase. In some embodiments, the subunit of either or both of the ATP citrate lyase genes (e.g., yariowiapolypical ATP citrate lyase; Genolevures YALI0D24431g and YALI0E34793g) Lt; / RTI > In certain embodiments, the activity of ATP citrate lyase in modified yeast is increased by the insertion and / or heterologous expression of an animal ATP lyase gene comprising a single subunit complete enzyme. In some preferred embodiments, the ATP citrate lyase in the genetically modified yeast is modified such that its activity and / or expression is increased when compared to the corresponding natural yeast. In certain preferred embodiments, the genetically altered yeast has at least 1.2-fold the activity and / or expression of the activity and / or expression of the ATP citrate lyase in the genetically modified yeast relative to the activity and / or expression of the native yeast; Or 1.5-fold, or 2-fold; Or 3-fold, or 4-fold; Or 5-fold; Or 10-fold, or 10-fold; Or 20-fold; Or 50-fold; Or 100-fold, or 1,000-fold; Or 10,000-fold, or 100,000-fold, or 1,000,000-fold higher.

In certain preferred embodiments of this aspect, the genetically modified yeast is a genetically modified yeast; In another preferred embodiment, the genetically modified yeast is a transgenic yeast. Additional embodiments are yeasts that include both transgenic and genetic modifications.

As used herein, the phrase "genetically modified yeast" refers to transgenic yeast or genetically altered yeast.

As used herein, the term "natural yeast" refers to a yeast that is not genetically modified (i.e., transgenically or genetically altered). Natural yeasts include not only wild-type yeast, but also optionally hybridized yeast so as to achieve certain characteristics.

The phrase "transgenic yeast" refers to a yeast having a gene obtained from another yeast species or non-yeast species. Such genes may be referred to as "transgene ".

As used herein, the term "target gene" refers to a gene encoding an enzyme to be modified.

The phrase "genetically modified yeast" refers to yeast with one or more genetic modifications, e.g., transgene containing one or more designed mutation (s) and / or modified enzyme. Through such a mutation, a modified enzyme having an activity different from that of a natural enzyme can be produced. Such differences may include differences in substrate specificity or activity levels. As used herein, "transgenic yeast" is a type of "genetically modified yeast. &Quot;

The phrase "maintainability yeast" refers to yeast that contains lipids that can be extracted from an organism at a cell dry weight (cdw) of at least about 20%. The ability to accumulate lipid levels to at least about 20% cdw is not limited to specific species; My process lipoic lipoic FER (Lipomyces lipofer), L. Le keyiyi star (L. starkeyi), L. Ruth tetrahydro spokes (L. tetrasporus), Candida Li poly urticae (Candida lipolytica), (C. diddensiae in C. diden cyano ), C. Lee poly para urticae (C. paralipolytica), C. Cuba other (C. curvata), Cryptococcal kusu Albi Douce (Cryptococcus albidus), Cryptococcal kusu Lau alkylene Chantilly (Cryptococcus laurentii), geo tree glutamicum alkanediyl Doom ( Geotrichum candidum ), Rhodotorula The strains of Rhodotorula graminus , Trichosporon pullans , Rhodophoridium toruloides , Rhodotorula glutinus , Rhodotorula gracilis , and Yarrowia polyrotica are strains of about 20 More than% cdw of lipid has been reported. See, for example, U.S. Patent No. 4,032,405 (Tatsumi et al.), And Rattray, Microbial Lipids, Vol. 1 (1998).

As used herein, the term "about" means ± 10% in terms of quantity. For example, about 3% "would include 2.7-3.3%, and" about 10% "would include 9-11%.

Unless otherwise indicated, all percentages referred to herein are% by weight.

Other features and advantages of the present invention will become apparent from the following preferred embodiments and claims.

DETAILED DESCRIPTION OF THE INVENTION

The increased amount of isoprenoid produced by the genetically modified yeast may be the result of mutating or transforming one or more enzymes contained in the isoprenoid biosynthetic pathway. For example, acetyl-CoA carboxylase (or "ACCase"), HMG-CoA reductase, squalene epoxidase, and squalene synthetase may be modified or mutated.

In a preferred embodiment, a genetically modified yeast expressing a modified enzyme is produced by introducing a mutation in the enzyme through the use of a gene repair oligonucleotide base as described herein. The method can be performed by any of a number of methods well known in the art (including, for example, microcarriers, microfibers, electroporation, electroporation, microinjection, LiOAc, gene chongbeop, Spain ropeul lasting, and / or Agrobacterium (Agrobacterium) (for example, in [McClelland, CM, Chang, YC, and Kwon-Chung, KJ (2005) Fungal Genetics and Biology 42 : 904-913)) into yeast cells, and identifying cells containing the mutated enzyme.

In one aspect of the invention there is provided an isoprenoid extracted from a genetically modified yeast disclosed herein. In a related aspect, squalene extracted from genetically modified yeast described herein is provided.

In another aspect of the present invention, there is provided a process for producing an isoprenoid, preferably squalene. In certain embodiments, the method comprises providing a genetically modified yeast as described herein, and extracting squalene from the yeast.

In yet another embodiment of this aspect of the invention there is provided an isoprenoid extracted from said genetically modified yeast or transgenic yeast.

Yeast type

The compositions and methods as disclosed herein may be based on any of a number of yeast species or strains. In certain embodiments, the yeast is a retainable yeast. For example, the yeast may be selected from the group consisting of Cryptococcus clavatus (e.g. ATCC 20508), Yarrowia polytyrica (e.g. ATCC 20688 or ATCC 90811), Rhodotorula glutinus (e.g. ATCC 10788 or ATCC 204091), and Rhodophoridium toruloides. The present inventors have found that Cryptococcus cuvatus and Rhodotorula glutinus grow at very high cell densities on a wide variety of substrates as compared to certain other yeast strains (for example, Yarrowia polytyrica) And that it produces a large amount of total lipid under culture conditions. Thus, in certain embodiments, Cryptococcus catus and Rhodotorula glutinus may be particularly advantageous for compositions and methods as disclosed herein. There are a number of genetic tools that are well developed, specific for the gall bladder polytyks (e.g., transformation protocols, selectable markers); In such cases, in some embodiments, yariowiopolytica may be particularly advantageous for compositions and methods as disclosed herein.

Gene repair oligonucleotide base

The present invention can be practiced by using a "gene repair oligonucleotide base" having the morphological and chemical properties as detailed below. "Gene repair oligonucleobase" of the present invention refers to a hybrid double oligonucleotide, a molecule containing a non-nucleotide, a single strand oligodeoxynucleotide, and other gene repair molecules described in the patents and patent publications mentioned below . The "gene repair oligonucleobase " of the present invention is also referred to as" oligonucleobase made by recombination " "RNA / DNA chimeric oligonucleotide "; "Chimeric oligonucleotides "; "Hybrid double oligonucleotide (MDON) "; "RNA DNA oligonucleotide (RDO) "; "Gene-targeted oligonucleotides ";"Genoplast"; "Single stranded oligonucleotide "; "Single stranded deoxynucleotide mutant vector "; "Double mutant vector"; And "heterologous mutant vectors ", as well as other names such as those described in the scientific and patent publications.

Having the morphology and chemical properties as described in U.S. Patent No. 5,565,350 (Kmiec (Crick I)) and U.S. Patent No. 5,731,181 (Crick (Crick II)) The oligonucleotide base is suitable for use as the "gene repair oligonucleotide base" of the present invention. The gene repair oligonucleotide bases in Crick I and / or Crick II include two complementary strands, one of which is a segment of at least one RNA-type nucleotide that forms a base pair with the remaining one strand DNA-type nucleotide (" RNA segment ") < / RTI >

It is disclosed that Chromium II can be substituted for non-nucleotides including purine and pyrimidine bases in place of nucleotides. Additional gene repair molecules that may be used in the present invention are described in U.S. Patent Nos. 5,756,325; 5,871,984; 5,760,012, 5,888,983, 5,795,972, 5,780,296; 5,945,339; 6,004,804; And 6,010,907 and International Patent No. PCT / US00 / 23457; And International Patent Publication Nos. WO 98/49350, WO 99/07865, WO 99/58723; WO 99/58702; And WO 99/40789, each of which is incorporated herein by reference in its entirety.

In one embodiment, the gene repair oligonucleobase can be obtained by replacing the 2'-hydroxyl with a fluoro, chloro or bromo functional group, or by placing a substituent on the 2'-O, Is a hybrid double oligonucleotide in which the nucleotide is resistant to RNase. Suitable substituents include the substituents taught by Chmick II. Alternative substituents include the substituents taught by U.S. Patent No. 5,334,711 (Sproat), and those disclosed in patent publications EP 629 387 and EP 679 657 (collectively Martin Applications) Incorporated herein by reference). As used herein, a 2'-fluoro, chloro or bromo derivative of a ribonucleotide, or a ribonucleotide in which the 2'-OH is substituted with a substituent as described in the Martin application or sprout, is referred to as a "2'- Quot; nucleotide ". As used herein, the term "RNA-type nucleotide" refers to a nucleotide sequence that is complementary to another nucleotide of a hybrid double oligonucleotide by either an unsubstituted phosphodiester linkage, or by non-natural bonds taught by & Substituted 2'-hydroxyl or 2'-substituted nucleotide. As used herein, the term "deoxyribonucleotide" refers to a nucleotide sequence encoding a gene repair oligonucleotide by either an unsubstituted phosphodiester linkage or by non-natural linkages taught by < RTI ID = Means a nucleotide having 2 ' -H, which can be linked to other nucleotides of the base.

In certain embodiments of the invention, the gene repair oligonucleobase is a hybrid double oligonucleotide linked only by an unsubstituted phosphodiester linkage. In an alternative embodiment, the bond is due to a substituted phosphodiester, a phosphodiester derivative, and a non-phosphorus-based bond, as taught by Chmick II. In yet another further embodiment, each RNA-type nucleotide in the mixed double oligonucleotide is a 2 ' -substituted nucleotide. Particular preferred embodiments of the 2'-substituted ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-propyloxy, 2'-allyloxy, 2'-hydroxylethyloxy, Hydroxyethyloxy, 2'-fluoropropyloxy and 2'-trifluoropropyloxy-substituted ribonucleotides. More preferred embodiments of 2'-substituted ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-methoxyethyloxy, and 2'-allyloxy substituted nucleotides. In another embodiment, the hybrid double oligonucleotide is linked by an unsubstituted phosphodiester linkage.

Although hybrid double oligonucleotides having only one type of 2'-substituted RNA-type nucleotide are more convenient for synthesis, the method of the present invention can be used by using mixed double oligonucleotides having two or more types of RNA-type nucleotides . The interruption caused by the introduction of deoxynucleotides between the two RNA-type trinucleotides may not affect the function of the RNA segment, and thus the RNA segment includes, for example, an "interrupted RNA segment" . An uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative embodiment, the RNA segments may alternatively comprise 2'-OH nucleotides that are resistant to RNase and unsubstituted 2'-OH nucleotides. Preferably, the hybrid double oligonucleotide has fewer than 100 nucleotides, more preferably less than 85 nucleotides, and more than 50 nucleotides. The first and second strands form a Watson-Crick base pair. In one embodiment, the strands of the hybrid double oligonucleotide are covalently joined by a linker, such as, for example, a single stranded hexa, penta or tetranucleotide, such that the first and second strands form a single 3 ' Terminus of a single oligonucleotide chain. The 3 'and 5' ends can be protected through the addition of a "hairpin cap" through which the 3 'and 5' terminal nucleotides form a Watson-Creek pair with adjacent nucleotides. Additionally, a second hairpin cap may be placed at the junction between the first and second strands remote from the 3 'and 5' ends, through which the Watson-cre pair formation between the first and second strands And stabilized.

The first and second strands include two sites that are identical to the two fragments of the target gene, i. E., Have the same sequence as the target gene. The homology region comprises the nucleotides of the RNA segments and may comprise one or more DNA type nucleotides consisting of linked DNA segments and may also include DNA type nucleotides not contained in the interposed DNA segment. Two homologous sites are separated by a region having a sequence that is different from the sequence of the target gene, called "heterologous site ", each of which is adjacent to the heterologous site. The heterologous site may comprise one, two, or three mismatched nucleotides. The mismatched nucleotides may be contiguous or, alternatively, may be separated by one or two nucleotides homologous to the target gene. Alternatively, the heterologous site may also include insertion of one, two, or three or no more than five nucleotides. Alternatively, the sequence of the hybrid double oligonucleotide may differ from the sequence of the target gene only by deletion of 1, 2, 3 or 5 nucleotides from the hybrid double oligonucleotide. In this case, the length and position of the heterologous site are considered to be the same as the deletion length, although no nucleotide of the hybrid double oligonucleotide is included in the heterologous site. The distance between the fragments of the complementary target gene in the two homology regions is equal to the length of the heterologous region in which substitutions or substitutions are intended. If the heterologous site comprises one insert, then the homologous site will be present in the mixed double oligonucleotide in a state where the complementary homologous fragment is separated farther than when the complementary homologous fragment is present in the gene and the heterologous site The opposite situation can be applied when coding deletion.

The RNA segment of the hybrid double oligonucleotide is a homologous site, i.e., each part of the same site as a fragment of the target gene in the sequence, which together preferably comprises at least 13 RNA-type nucleotides, preferably 16-25 Or, more preferably, 18-22 RNA-type nucleotides, or most preferably 20 nucleotides. In one embodiment, the RNA segments of the homology region are separated by and are adjacent to the intervening DNA segment, i. E., "Linked" by intervening DNA segments. In one embodiment, each nucleotide of the heterologous site is a nucleotide of an intervening DNA segment. An intervening DNA segment comprising a heterologous site of a hybrid double oligonucleotide is termed a "mutator segment ".

In another embodiment of the present invention, the gene repair oligonucleotide base is a single stranded deoxynucleotide mutant vector (SSOMV), which is disclosed in International Patent Application PCT / US00 / 23457, U.S. Patent Nos. 6,271,360, 6,479,292, And 7,060,500 (the entire contents of which are incorporated herein by reference). Sequences of SSOMV are disclosed in U.S. Patent Nos. 5,756,325 and 5,871,984; 5,760,012, 5,888,983, 5,795,972, 5,780,296, 5,945,339, 6,004,804, and 6,010,907, and International Publication No. WO 98/49350; WO 99/07865; WO 99/58723; ≪ / RTI > WO 99/58702, and WO 99/40789. The sequence of SSOMV comprises two sites, homologous to the target sequence, separated by a site containing the desired genetic alteration, referred to as the mutator site. The mutator site may have another sequence, but may have a sequence having the same length as the sequence that separates the homologous site in the target sequence. Such mutator sites may cause substitution. Alternatively, the homologous sites in the SSOMV may be adjacent to each other, while the homologous sites in the target sequence having the same sequence are separated by one or more nucleotides. The SSOMV causes deletion from the target gene of the nucleotide that does not exist in SSOMV. Finally, the sequence of the same target gene as the homology region may be contiguous in the target gene, but may be separated by one or more nucleotides in the SSOMV sequence. Such an SSOMV induces one insertion into the sequence of the target gene.

The nucleotides of SSOMV are only deoxyribonucleotides linked by unmodified phosphodiester linkages, except for the linkage between the 3 ' and / or 5 ' terminal nucleotides, or alternatively two 3 ' The bond between the terminal nucleotides may be a phosphorothioate or a phosphoamidate. As used herein, the bond between nucleotides is a bond between the nucleotides of SSOMV, which does not include a bond between the 3 'terminal nucleotide or the 5' terminal nucleotide and the blocking substituent (see above). In certain embodiments, the length of the SSOMV is from 21 to 55 deoxynucleotides, so the length of the homology region is the total length consisting of at least 20 deoxynucleotides, and at least two homologous sites each comprise at least eight Lt; RTI ID = 0.0 > and / or < / RTI >

SSOMV may be designed to be complementary to either the coding or non-coding strand of the target gene. When the desired mutation is a substitution of a single base, it is preferred that both the mutator nucleotides are pyrimidines. It is preferred that the mutator nucleotides, and the targeted nucleotides in the complementary strands, are both pyrimidines to the extent that they are consistent with achieving the desired functional result. SSOMV coding for a base-translational mutation, SSOMV, in which C or T mutator nucleotides are mismatched with C or T nucleotides, respectively, in a complementary strand are particularly preferred.

In addition to oligodeoxynucleotides, SSOMV may comprise a 5 'blocking substituent attached to the 5' terminal carbon through the linker. The chemical nature of the linker is important only in its length, it must be at least six atoms in length, and the linker must be flexible. A variety of non-toxic substituents, such as biotin, cholesterol or other steroid or non-intercalating cationic fluorescent dyes, may be used. Reagents commercially available as Cy3 (TM) and Cy5 (TM) by Glen Research, Stirling, Va., Are particularly preferred as reagents for the preparation of SSOMVs, which, when introduced into oligonucleotides, 3'-tetramethyl N, N'-isopropyl substituted indomonocarbocyanine and blocked diborothiocyanine dyes. Cy3 is most preferable. When the indolocarbocyanine is N-oxyalkyl substituted, it can be easily linked to the 5'end of the oligodeoxynucleotide via a phosphodiester having a 5'terminal phosphate. The chemical nature of the dye linker between the dye and the oligodeoxynucleotide is not critical and is chosen for convenience in synthesis. When a commercially available Cy3 phosphoramidite is used as directed, the resulting 5'-modification is a combination of a blocking substituent with a N-hydroxypropyl, N'-phosphopathadyldipropyl 3,3,3 ', 3 '- tetramethylindo monocarbocyanine linker.

In a preferred embodiment, the indcarbocyanine dye is tetrasubstituted at the 3 ' and 3 ' positions of the indole ring. Without being bound by theory, such substitution prevents the dye from becoming an intercalate dye. The identity of substitution at this position is not important. In addition, SSOMV may have a 3 'blocking substituent. In addition, the chemical nature of the 3 'blocking substituent is not critical.

Heterogeneous expression

In certain embodiments, heterologous expression is used to express an exogenous gene, or an additional copy of an endogenous gene, in a yeast (e.g., yariowiropolytica). The heterologous expression in yeast can be performed by using methods well known in the art. Expression of foreign genes, or additional copies of endogenous genes, in yeast using heterologous expression includes (a) a promoter sequence for transcription initiation, (b) a terminator sequence for transcription termination, and (c) a selectable marker Lt; / RTI > vector. The heterologous expression and expression vectors are described in, for example, Madzak, C., Gaillardin, C., and Beckerich, JM., 2004 Heterologous Protein Expression and Secretion in the Non-Conventional Yeast Yarrowia lipolytica : a review, Journal of Biotechnology 109 : 63-81]. A non-limiting list of selectable marker genes that may be used include E. coli hph encoding ura3 , lys5 , trp1 , leu2 , ade1 , hygromycin resistance, and Saccharomyces < SUC2 from S. cerevisiae . A non-limiting list of promoters that may be used includes pLEU2, pXPR2, pPOX2, pPOT1, pICL1, pG3P, pMTP, pTEF, and pRPS7. A non-limiting list of terminator sequences that may be used includes XPR2t , LIP2t , and PHO5t .

In certain embodiments, the Yarrow subtotal Li poly Mathematica is used LYS1 (halogeno Levu less YALI0B15444g), TRP1 (halogeno Levu YALI0B07667g less), and ADE1 (halogeno Levu less YALI0E33033g) as a selectable marker at least one gene.

In a particular embodiment, the integrated expression vector comprises a Yarrow subtotal Li poly urticae its route gene, alkane or glycerol using gene ACC1, HMG1, ERG1, and at least one promoter and is selected from the group consisting of ERG9 / or termination factor sequence .

In certain embodiments, subunits of one or both of the Yarrowia polytitica ATP citrate lyase (GenoRevresses YALI0D24431g and YAL10E34793g) are overexpressed in Yarrowia polyrotica.

Modified enzyme

A gene encoding an enzyme involved in the fatty acid biosynthetic pathway and the isoprenoid biosynthetic pathway is a desirable target for mutation. In some embodiments, the target gene encodes an acyl CoA carboxylase. In another embodiment, the target gene encodes an HMG-CoA reductase. In another embodiment, the target gene encodes a squalene epoxidase. In another embodiment, the target gene encodes a squalene synthase. In certain embodiments, the target gene encodes an ATP citrate lyase. Mutations may be devised that reduce or eliminate the activity of the enzyme, enhance the activity of the enzyme, or alter the activity of the enzyme (e.g., substrate selectivity changes).

In wild-type fat-retaining yeast, acetyl-CoA is widely distributed via fatty acid biosynthesis through acetyl-CoA carboxylase (ACCase). Therefore, it may be desirable to decrease the enzyme expression or specific activity of ACCase in order to increase the amount of acetyl-CoA available for squalene synthesis. An exemplary gene sequence for ACCase is the ACC1 gene of Saccharomyces cerevisiae, identified as Accession No. Z71631. Thus, in certain embodiments, the decrease in the intracellular activity of ACCase, an enzyme at the junction between mevalonate biosynthesis and triglyceride biosynthesis, will reduce the amount of acetyl-CoA distributed for oil synthesis, His availability will increase.

The HMG-CoA reductase activity is an enzyme whose rate is limited to isoprene biosynthesis. Exemplary gene sequences for the HMG-CoA reductase include HMGl and HMGl genes of Saccharomyces cerevisiae, respectively, as Accession Nos. NC_001145 and NC_001144. Thus, in certain embodiments, the HMG-CoA reductase activity will be increased by modifying the HMGR gene to increase transcription and / or stabilize the resulting protein and / or decrease product feedback inhibition.

Reducing ACCase activity in yeast and / or increasing HMG-CoA reductase activity can result in the production of important isoprenoid-producing organisms capable of producing a large number of related isoprenoid products by manipulation of subsequent enzymes in the pathway.

The squalene epoxidase catalyzes the first critical step of sterol biosynthesis. An exemplary gene sequence for squalene epoxidase is the ERG1 gene of Saccharomyces cerevisiae, denoted accession number NC_001139. Thus, in certain embodiments, the squalene epoxidase activity and / or expression will be attenuated in the yeast by, for example, residues important in catalysis in the amino acid sequence of the enzyme.

Squalene synthase catalyzes the synthesis of squalene, which condenses two c15 isoprene precursors (paruncyldiphosphate (FPP)). An exemplary gene sequence for squalene synthase is the ERG9 gene of Saccharomyces cerevisiae, as identified by accession number NC_001140. Thus, in certain embodiments, squalene synthase activity and / or expression will be increased in yeast.

ATP citrate lyase (EC 4.1.3.8) catalytically cleaves citrate to produce acetyl CoA and oxaloacetate. Acetyl CoA can be used by ACCase for fatty acid biosynthesis or by acetyl CoA acetyltransferase for the production of isoprenes and derivatives, such as squalene.

As a result, metabolic alteration will result in the transfer of carbon from acetyl-CoA to squalene, a major competing route to this carbon flow will be weakened, and the production of squalene will increase significantly.

Gene repair oligonucleotide transfer to yeast cells

Any of the methods commonly known in the methods of the present invention for transforming yeast cells with gene repair oligonucleotides can be used. Exemplary methods include by microcarrier or microfiber use, microinjection, electroporation, LiOAc, gene gun techniques, spiroplastination, and / or Agrobacterium (see, for example, McClelland, CM, Chang , YC, and Kwon-Chung, KJ (2005) Fungal Genetics and Biology 42: 904-913).

In some embodiments, metallic microcarriers (microspheres) are used to introduce large DNA fragments into enzyme cell cells with cell walls by propellant permeation (gene gun delivery), which is well known to those skilled in the art. A general technique for selecting microcarriers and launch devices therefor is described in U.S. Patent Nos. 4,945,050; 5, 100, 792 and 5,204, 253.

Specific conditions for the use of microcarriers in the method of the present invention are described in International Publication No. WO 99/07865, US09 / 129,298. For example, ice-cold microcarriers (60 mg / mL), mixed double oligonucleotides (60 mg / mL), 2.5 M CaCl ₂ and 0.1 M sphamidine were added in order; The mixture is gently agitated, for example, for 10 minutes, allowed to stand at room temperature for 10 minutes, after which the microcarrier is diluted with 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol. An example of the concentrations of the liquid components of the attachment is raised 8-10 ㎍ / ㎕ microcarrier, 14-17 ㎍ / ㎕ mixed duplex comprises a nucleotide, 1.1-1.4 M CaCl ₂ and 18-22 mM spermidine. In one example, the concentration of the component is 8 μg / μl microcarrier, 16.5 μg / μl mixed double oligonucleotide, 1.3 M CaCl ₂ and 21 mM spermidine.

Gene repair oligonucleotide bases can also be introduced into yeast cells for the practice of the invention by using microfibers that penetrate cell walls and cell membranes. U.S. Patent No. 5,302,523 (Coffee et al.) Describes the use of 30 x 0.5 urn to 10 x 0.3 탆 silicon carbide fibers to promote the transformation of maize suspension cultures of Black Mexican Sweet . Any mechanical technique that can be used to introduce DNA for transformation of yeast cells using microfibers can be used to deliver the gene repair oligonucleotide base.

An example of delivering a gene repair oligonucleotide base using a microfiber is as follows. Sterile microfibers (2 [mu] g) are suspended in 150 [mu] l of yeast growth medium containing about 10 [mu] g of mixed double oligonucleotide. The yeast culture is submerged and an equal volume of packed cells and a sterile fiber / nucleotide suspension is vortexed and plated for 10 minutes. Immediately or after a period of up to about 120 hours, the selection medium is added to suit a particular trait.

In an alternative embodiment, the gene repair oligonucleotide base can be delivered to the yeast cell by electroporation of the protoplast derived from the yeast cell. Protoplasts are formed by treating yeast cells with enzymes according to techniques well known to those skilled in the art (see for example Gallois et al., 1996, in Methods in Molecular Biology 55: 89-107, Humana Press, Totowa , NJ, Kipp et al., 1999, in Methods in Molecular Biology 133: 213-221, Humana Press, Totowa, NJ). Protoplasts do not need to be cultured in growth medium prior to electroporation. Exemplary conditions for electroporation are the presence of 3 x 10 ⁵ protoplasts in 0.3 mL total volume with a concentration of the gene repair oligonucleotide base ranging from 0.6-4 / / mL.

In yet another alternative embodiment, the gene repair oligonucleotide base can be delivered to the yeast cell by whisker or microinjection of yeast cells. The so-called whisker technique is essentially performed as described in Frame et al., 1994, Plant J. 6: 941-948. Gene repair oligonucleotide bases are added to whiskers and used to transform yeast cells. The gene repair oligonucleotide base may be co-incubated with a plasmid comprising a sequence encoding a protein capable of forming a recombinant and / or a gene repair complex in yeast cells, whereby the target sequence in the target gene and Restoration of the gene between oligonucleotides is promoted.

Screening of enzymes with the desired modified enzyme

The yeast expressing the modified enzyme can be identified by any of a number of means. In one approach, co-transformation strategies are used to target both selectable transitions (i. E., Markers) and non-selectable transitions (e. G., Target genes of interest) in the same experiment to gene repair oligonucleobases (GRON) is used. In this way, cells that have not been delivered GRON, or that can not deliver a transition where the GRON is embodied by RON will be removed. The transmission of GRON targeting unrelated genes is not predicted to be selective and it can be predicted that a colony with a transition selected to some degree frequently can also have a conversion to one of the other target genes. The conversion event will be analyzed by a single nucleotide polymorphism (SNP) analysis.

Thus, genomic DNA is extracted from yeast and individual DNA samples are screened for each target using SNP detection techniques, such as allele-specific polymerase chain reaction (ASPCR). In order to independently ascertain sequence changes in the positive yeast, the appropriate region of the target gene can be PCR amplified, the sequence of the resulting amplicon can be directly analyzed, the amplicon can be cloned, and the sequence of multiple inserts can be analyzed have.

Alternatively, mutagenic incorporation into the gene of interest can be achieved by a number of molecular biology techniques (e. G., Amplification methods, e. G., PCR and single nucleotide primer extension Analysis method). Larger mutations can be detected by amplification and sequencing of the target gene region to be mutated.

Alternatively, yeast or yeast cells containing the modified enzyme can be identified, for example, by analysis of an isoprenoid composition produced by yeast. Thus, the yeast can be grown, the oil extracted, and analyzed using methods known in the art (e.g., gas chromatography)

Example

Example 1. Cryptococcus cuvatus and Rhodotorula glutinosa Transformation system

A screenable marker for transforming the strain from kanamycin susceptible to resistant to produce Cryptococcus cuvatus (ATCC strain 20508) and Rhodotorula glutinus (ATCC strains 10788 and 204091) A KANMX expression cassette (promoter-gene-terminator) was used that confers kanamycin resistance to S. cerevisiae (see, for example, Baudin, A., et al. (1993) Nucleic Acids Research (21) 3329-3330). Not only did the strain transformed into the expression cassette, but also plasmids reported in R. glutinus (including the DNA replication origin) (see, for example, Oloke. JK, and Glick, BR Journal of Biotechnology 5 (4): 327-332)). Electrophoresis, LiOAc, genetic gun technique, spiropalatability, and / or Agrobacterium (e.g., McClelland, CM, Chang, YC, and Kwon-Chung, KJ (2005) Fungal Genetics and Biology 42: 913]) introduced DNA into C. curvatus and R. glutinus .

Example 2. Selectable markers

To produce uracil nutrient requiring mutants in Cryptococcus cuvatus and Rhodotorula glutinus, cells are grown in a minimal medium containing the anti-metabolite 5-fluororotic acid to produce lesions in the ura3 or ura5 gene Resistant mutants. 33, Cryptococcal kusu Cuba and stored the tooth stable 5-FOA ^R colonies and 20, torulra article Ruti Taunus stable 5-FOA ^R colonies aberration view. The wild-type URA3 gene from Cryptococcus cuvatus and Rhodotorula glutinus was cloned and the sequence of the mutant ura3 gene in the 5-FOA ^R isolate was analyzed.

Other nutritional requirement markers were cloned by functional complementation in Saccharomyces cerevisiae (Ho, YR, and Chang, MC (1988) Chinese Journal of Microbiology and Immunology 21 (1): 1-8) ). Genomic and / or cDNA libraries were prepared from Cryptococcus cuvatus and Rhodotorula glutinus to ligate into uracil-selectable Saccharomyces expression vector for transformation into strain YPH500 ( MATα ura3-52 lys2- 801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1 ) lysine, adenine, tryptophan, histidine, and leucine precursors . The sequences of the corresponding genes for LYS2, ADE2, TRP1, HIS3, and LEU2 from these raw nutrients were analyzed from genomic or cDNA inserts.

Example 2. Genetic manipulation in yeast using RTDS technology

The leu2, lys5 and ura3 gene alleles (leu2-35 lys5-12 ura3-18 XPR2B) were cloned by PCR from Yarrowia polytrica strain ATCC 90811 and these sequences were compared with the wild type allele to confirm the difference Respectively.

In the case of ura3, the difference is in position 1365 (A → G mutation, thereby forming a silent mutation from AAA to AAG (AAA → AAG) encoding lysine), position 1503 (AAGAA additional sequence at ATCC 90811, (Additional T in ATCC 90811), and 1978 (SEQ ID NO: 10), but the sequence is returned to YL URA3 in GenBank) (C → T mutation, forming a terminating mutation that cleaves a protein consisting of 24 amino acids without carboxy terminus). A GRON oligonucleotide was designed to convert the STOP (TGA)? R (CGA) to recover the raw nutrients, thereby obtaining 264R based on the YlUra3-YLU40564 amino acid numbering. The GRON used was YlUra31264 / C / 40 / 5'Cy3 / 3'idC

(Which has the sequence VCGAGGTCTGTACGGCCAGAACCGAGATCCTATTGAGGAGGH), and YlUra31264 / NC / 40 / 5'Cy3 / 3'idC

(Which has the sequence VCCTCCTCAATAGGATCTCGGTTCTGGCCGTACAGACCTCGH, where V = CY3; H = 3'DMT dC CPG). 10, 30, and 50 쨉 g of each GRON were transformed with Yarrowia polytrica strain ATCC 90811 using the lithium acetate-based method and plated on ura-2% glucose. It was found that a total of 82 ura + colonies with GRON designed using coding strand and 6 colonies with GRON designed using non-coding strand were obtained, whereby strand misalignment was common in transforming into gap-repair oligonucleotides Proven. Sequence analysis of 18 coding-stranded transfectants proved that the intended variation in 17 clones occurred.

In the case of LEU2, the difference is at position 1710 (no further C exists, resulting in a frame shift and premature protein termination); Position 1896 (additional T); Position 2036 (T? A mutation, located after the stop codon); It was found at position 2177 (additional T in the defect, located after the stop codon).

For LEU2, the difference is position 1092 (G? A TCG? TCA, conservative substitution (serine)); Position 1278 (G? A CAG? CAA, conservative substitution (glutamine)); It was found at position 1279 (G → A GGT → ATT, V to I (V → I)).

Thus, for example, mutations may be used for a variety of purposes, such as conversion of a raw nutritive yeast to become an auxotrophic yeast, and vice versa.

Similar strategies were performed to demonstrate the efficacy of RTDS technology in Cryptococcus cuvatus and Rhodotorula glutinus, as described for Yarrowioli Polytica by calibrating the ura3 mutation for restoration to the raw nutrient .

Example 3. Target gene cloning

Sequences for ACCase, HMGR, squalene synthase and squalene epoxidase available from the NCBI database, originating from Saccharomyces and other yeasts, are used as the source of the PCR primers and the corresponding gene is amplified using its corresponding regulatory region (Promoter, terminator) from Cryptococcus cuvatus and Rhodotorula glutinus. To identify 'up' and 'down' promoter mutations, respectively, that increase or decrease transcription, the promoters for the four genes are cloned together with a relatively error-prone DNA polymerase, And these fragments were cloned into a plasmid fused with a green fluorescent protein (GFP) or beta-galactosidase reporter gene to perform an in vitro test against S. cerevisiae or E. coli. The promoter "upstream" mutation was reintroduced into the HMGR and squalene synthase genomic sequences by RTDS, while the "downstream" promoter mutation proceeded in the genomic ACCase and squalene epoxidase sequences. Promoters from essential genes (e.g., GAPDH, actin) in R. glutinus and C. clavatus were cloned using heterologous gene expression. The primers for PCR cloning were designed to be homologous to the gene in S. seriginosa.

Example 4. Target gene manipulation for increased squalene production

ACCase . The copy number of the ACCase gene was measured in R. glutinus and C. clavatus, and other yeast. RTDS was used to reduce ACCase expression by introducing a stop codon immediately after the translation start site in any additional copies. Correlation between ACCase mRNA levels and ACCase enzyme specific activities in S. sakazai serebaciae has been demonstrated.

Squalene epoxidase . Similarly, the accumulation of squalene in S. cerevisiae was increased by destroying one squalene epoxidase copy in the diploid. Kamimura, N., Hidaka, M., Masaki, H., and Uozumi, T. (1994) Appl. Microb. Biotech. 42: 353-357]. The number of copies of squalene epoxidase in R. glutinus and C. clavatus and other yeast was measured and RTDS was used to form or insert a stop codon immediately after the translation start site in the additional copies except the first.

HMGR . The HMGR enzyme, both saccharomyces cerevisiae and mammalian, contains an amino acid sequence at its linker site that is present in a number of short-lived proteins that undergo rapid intracellular conversion in eukaryotic cells (Rogers, S (1986) Science 234 : 364-368], and [Chun, KT, and Simoni, RD (1991) J. Biol. Chem. 267 (6): 4236-4246 ]) Reference). When present, similar sequences were identified in the HMGR gene of R. glutinus and C. clavatus and the HMGR protein conversion was reduced by removing it using RTDS. Such similar sequences have also been found in the S. cerevisiae squalene synthase gene and also whether the sequence is also present in the squalene synthase gene of R. glutinus and C. clavatus. R. glutinus and C. clavatus squalene synthetase also reduced protein conversion by removing the sequence using RTDS.

S. cerevisiae HMGR contains two highly conserved domains, of which the N-terminal 552 amino acids are responsible for membrane binding. Overexpression of truncated HMG1 protein containing only the catalytic portion of the C-terminus increased the HMG-CoA activity 40-fold in S. seriginosa and increased the accumulation of squalene to 5.5% dry matter (Polakowski , T., Stahl, U., and Lang, C. (1998) Appl. Microbiol. Biotech. 49: 66-71). R. glutinus and C. clavatus HMGR proteins have similar structures and, if so, only the soluble catalytic domain was expressed using RTDS.

The protein structure and DNA sequences of HMGR are highly conserved between eukaryotic cells, from fungi to mammals, including membrane-bound N-terminal domains and catalytic C-terminal domains. The boundary between the two domain, may be hydrophobic plot of the transition region that is to be hydrophilic from hydrophobic, Yarrow subtotal Li poly urticae HMG1 gene map of the amino acid 500-600 region of the (halogeno Levu less Yarrow subtotal Li poly YALI0E04807g urticae) Chemistry . Yarrow subtotal Li poly hydrophobicity plot of Mathematica HMG1, and his, my process three Levy jiae (described as a truncated Saccharomyces [Donald, KAG, et al, 1997. Appl Environ Micro 63 (9):.... 3341-3344] ) And Candida utilis (Shimada, H. et al., 1998. Appl. Environ. Micro. 64 (7): 2676-2680)) to evaluate the homology to the N-terminus of the protein Residues 548 to 544 were selected. Thus, in one example, Yarrow subtotal Li poly urticae were expressed amino acids 548-1000 constituting the C- terminal domain of HMG1 I; The second example, the amino acids 544-1000 was expressed constituting the C- terminal domain of Yarrow subtotal Li poly urticae HMG1 I. A related example, Yarrow subtotal Li poly urticae reduce or expressing the 543-1000 amino acid constituting the C- terminal domain of HMG1 I; Or to express a Yarrow subtotal Li poly amino acids 545-1000 urticae constituting the C- terminal domain of HMG1, or I; Or to express a Yarrow subtotal Li poly amino acids 546-1000 urticae constituting the C- terminal domain of HMG1, or I; Or to express a Yarrow subtotal Li poly amino acids 547-1000 urticae constituting the C- terminal domain of HMG1, or I; Or it was expressed amino acids 549-1000 constituting the C- terminal domain of Yarrow subtotal Li poly urticae HMG1 I.

In Syrian hamsters, the activity of the HMGR catalytic domain is down-regulated by phosphorylation by AMP-dependent kinase (Omkumar, RV, Darnay, BG, and Rodwell, VW (1994) J. Biol. Chem. 269: 6810- 6814]) and S. seriginosa in a similar manner. It was determined whether the HMGR protein was similarly regulated in R. glutinus, C. clavatus, and other yeast, and if so, the phosphorylated site was removed using RTDS.

Squalene synthase . In mammalian systems, squalene synthetase is regulated by SREBP (sterol regulatory factor binding protein) to an equivalent level with the HMG-CoA synthase and parunaceptide diphosphate synthase (Szkopinsda, A., Swiezewska, E. et al. , and Karst, F (2000) Biochem. Biophys. Res. Comm. 267: 473-477). SREBP exists in three forms, one of which binds to the squalene synthase promoter. Determining whether such transcription factors and / or binding sites are present on the squalene synthase promoter of R. glutinus, C. clavatus, and other yeast, and, if present, determining whether squalene synthase Transcription factor and / or binding site promoting transcription.

Example 5. Growth conditions for Cryptococcus cactus

By determining the growth of Cryptococcus cactus, the optimal carbon source that could maximize its cell mass in the culture was determined. Among the rich media (10 g / L yeast extract, 20 g / L peptone) based on yeast extract, C. clavatus grew well in 2-20% w / v glucose, resulting in an increase in glucose over 16% w / v glucose The maximum level of 55 g / L cell dry weight (CDW) was reached. Similarly, C. clavatus grew in the same medium containing 3-12% w / v glycerol and reached CDW of 40 g / L in 12% w / v glycerol after 5 days. C. Cubatus also reached 23 g / L CDW, growing below 3.5% w / v in biodiesel glycerol (Imperial Western Products, Cochella, Calif.).

Example 6. Environmental manipulation of target genes for increased squalene production

Environmental manipulations were tested to increase the total yield of squalene. (A) inhibiting ACCase expression and / or activity using oleic acid, olive or other vegetable oil (s), inositol, choline, soraphen, fluazepop, and clotodim or other ACCase inhibiting herbicides, (b) inhibiting squalene epoxidase expression and / or activity using terbinafine, tolnaphthate and ergosterol or other squalene epoxidase inhibiting fungicides, (c) inhibiting the expression of squalene epoxidase in a glycerol-based medium (D) varying the source of nitrogen in the medium (organic vs. inorganic versus monolith / complex), (e) carbon addition method, (F) examining the effect of nutrient depletion other than the carbon source, (g) determining the effect of nutrient depletion on the sugar, sugar alcohol, alcohol, polyalcohols, and organic acids The mixture (H) selecting for growth on inhibitors of HMGR-inhibiting compounds, such as lovastatin or other statins, and (i) using lipophilic dyes or colorants To screen for high oil production in the culture and / or to analyze lipids that can be extracted using, for example, gravity or gas chromatography methods.

For example, Yarrowia polytilica ATCC 90904 was cultured in a medium with high carbon / nitrogen ratio (C / N = 420, literature 2006, "Optimized Culture Medium and Fermentation Conditions for Lipid Production by Rhodosporidium toruloides" Chinese Journal of Biotechnology 22 (4): 650-656) (Li, YH., Liu, B., Zhao, ZB., And Bai, Hereinafter "CYM001 medium"). When terbinafine concentration was above 12.5 ㎍ / ml, squalene in total lipids increased to 18.5%.

In another example, Yarrowia polyethica ATCC 90904 was cultured in CYM001 medium supplemented with 0-50 ug / ml oleic acid at 300 rpm at 30 캜 for 120 hours. It was found that the lipid accumulation was improved to 63.3% lipid / CDW (cell dry weight) when supplemented with 10 μl / ml oleic acid.

In a further example, Yarrowia polytilica ATCC 90904 was cultured in CYM001 medium supplemented with 0 to 200 μM of cleptomycin at 300 ° C and 300 rpm for 120 hours. When supplemented with 200 μM of clostom, the yield (mg) of squalene per 60-ml flask increased by 60-fold.

Increased oxygen induced differentiation of HMG1 and HMG2 in S. seriginosa, which resulted in rapid degradation of HMG2 under aerobic conditions and increased expression of HMG1 (Casey, WM, Keesler, GA, Parks, LW (1992) J. Bact. 174: 7283-7288). Whether the number of HMGR genes in the retentive yeast of the present invention is affected by oxygen and, if so, its expression and activity can be manipulated in a fermenter by changing the oxygen level.

Starting from "CYM001 medium" (Li, YH., Liu, B., Zhao, ZB., And Bai, FW. (2006) Chinese Journal of Biotechnology 22 (4): 650-656) Cell growth, total lipid content (%) / cell unit mass, and squalene (%) / total lipid were improved by changing the concentration of the components (including adding new components). The media components evaluated were carbon source: glycerol, glucose, nitrogen source: ammonium compound, nitrate, amino acid, inorganic salt: potassium, magnesium, sodium, iron, manganese, zinc, calcium, copper, yeast extract, lipid precursor and lipid synthesis Action factors: terbinafine, clestodim, oleic acid, palmitoleic acid, linoleic acid, linolenic acid and defoamer. Other factors assessed include inoculum (%), elapsed fermentation time, temperature, pH, back pressure, DO (DO), composition of nutrient source, nutrition strategy and agitation strategy

Example 7. Strain Selection

Conventional strain screening methods were used to increase his total squalene productivity in retentive yeast. Mutants induced by UV, nitroso guanidine, or ethane methyl sulfonate were screened and / or screened for increased squalene accumulation. It is also possible to apply a repetitive selection pressure on the strain, for example, a culture containing YEP (15 g / L yeast extract, 5 g / L peptone) medium containing 3% glycerol or lovastatin and other known HMGR inhibitors And repeatedly inoculated on the medium. In addition, repeated inoculation of the strain on CYM001 medium containing various amounts of glycerol and / or glucose, or on a medium containing lovastatin and / or other known HMGR inhibitors, and / or squalene synthetase inhibitors, / RTI > and / or squalene synthase activity is increased. Such mutations may be present in HMGR, squalene synthase, or other genes ("secondary site mutations").

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The invention illustratively described herein is not specifically disclosed in this application, but may be suitably performed under conditions where there are no elements or elements, limitations or limitations. Thus, for example, the words "comprising, including, containing, etc. " should be read broadly without limitation. Additionally, terms and expressions used herein are used as a tool to illustrate, It is to be understood that the invention is not intended to exclude any equivalents of the features disclosed and described in the use of the terms and expressions and may be variously modified within the scope of the claimed invention.

Thus, although the present invention has been specifically disclosed by preferred embodiments and certain features, it will be appreciated that variations, improvements and modifications of the present invention as disclosed herein and embodied therein may be reclassified by those skilled in the art, It is to be understood that such variations, modifications, and alterations are considered to be included within the scope of the present invention. The materials, methods, and embodiments provided are representative of the preferred embodiments, are illustrative and are not intended to limit the scope of the invention.

The present invention has been extensively and generally described herein. BRIEF DESCRIPTION OF THE DRAWINGS A more complete range of species and subspecies, each of which is included in the generic disclosure, also form part of the present invention. The foregoing includes a general description of the present invention provided that it is voiced on condition that any content is excluded from the above type regardless of whether the deleted material is specifically cited herein.

Further, in addition to the features or aspects of the present invention described in the Markush family, those skilled in the art will also recognize that the invention may also be described as a subset of any individual constituent or constituent constituting a Makush family by the above I will understand.

All publications, patent applications, patents, and other references mentioned herein are hereby expressly incorporated by reference in their entirety to the same extent as if each reference is individually incorporated herein by reference. Where there is a conflict of opinion, the present specification, including the definitions, will control.

Claims (10)

Wherein the genetically altered yeast is selected from the group consisting of (i) a modified endogenous squalene synthase gene mutated by a gene repair oligonucleotide base, or (ii) a modified endogenous squalene synthase gene, Wherein the genetically modified yeast comprises an exogenous squalene synthase gene and produces an increased amount of squalene compared to a yeast without mutation in the endogenous squalene synthase gene or a yeast without an exogenous squalene synthase gene. Comprising increasing or decreasing the activity or expression of squalene synthase,
Wherein the activity or expression of said enzyme is increased or decreased by (i) one or more designed mutations in an endogenous squalene synthase gene or (ii) introduction of an exogenous squalene synthase gene,
Wherein at least one of said one or more designed mutations is mutated by a gene repair oligonucleotide base and said at least one designed mutation is present at a designated position in said enzyme.
A method of producing squalene by genetically altered yeast that produces increased amounts of squalene compared to natural yeast. The composition of claim 1, wherein the yeast is an oleaginous yeast. The method according to claim 3, wherein the yeast is selected from the group consisting of Lipomyces lipofer , L. starkeyi , L. tetrasporus , Candida lipolytica , in C. diden cyano (C. diddensiae), C. Lee poly para urticae (C. paralipolytica), C. Cuba other (C. curvata), Cryptococcal kusu Albi Douce (Cryptococcus albidus), Cryptococcal kusu Lau alkylene Chantilly ( Cryptococcus laurentii ), Geotrichum candidum , Rhodotorula Such as Rhodotorula graminus , Trichosporon pullans , Rhodosporidium toruloides , Rhodotorula glutinus , Rhodotorula gracilis , and the like. Wherein the composition is selected from the group consisting of Yarrowia lipolytica . The composition of claim 1, wherein the yeast is not Yarrowia lipolytica . 3. The composition of claim 1, wherein said genetically modified yeast produces squalene more than twice as much as yeast expressing squalene synthase without one or more mutations or yeast without an exogenous squalene synthase gene. 3. The method of claim 2, wherein the yeast is oleaginous yeast. The method according to claim 7, wherein the yeast is selected from the group consisting of Lipomyces lipofer , L. starkeyi , L. tetrasporus , Candida lipolytica , in C. diden cyano (C. diddensiae), C. Lee poly para urticae (C. paralipolytica), C. Cuba other (C. curvata), Cryptococcal kusu Albi Douce (Cryptococcus albidus), Cryptococcal kusu Lau alkylene Chantilly ( Cryptococcus laurentii ), Geotrichum candidum , Rhodotorula Such as Rhodotorula graminus , Trichosporon pullans , Rhodosporidium toruloides , Rhodotorula glutinus , Rhodotorula gracilis , and the like. Wherein said composition is selected from the group consisting of Yarrowia lipolytica . 3. The method of claim 2, wherein the yeast is not Yarrowia lipolytica . 3. The method of claim 2, further comprising extracting squalene from the genetically modified yeast.